1. Field of the Invention
The present invention relates generally to a biosensor that can be used for the quantification of a specific component or analyte in a liquid sample. Particularly, this invention relates to a new and improved biosensor and to a new and improved method of fabricating a biosensor for the quantification of a specific component or analyte in a liquid sample such as creatinine, creatine, glucose, cholesterol, urea and the like. More particularly, this invention relates to a disposable biosensor that is inexpensive to manufacture. Even more particularly, this invention relates to a disposable biosensor and method that accurately measures various analytes such as creatinine, creatine, glucose, cholesterol and the like in small volume biological fluid samples. Still even more particularly, this invention relates to a method of measuring the concentration of various analytes in small volume biological fluid samples using a redox mediator and at least an enzyme based on the electrochemical mechanism.
2. Description of the Prior Art
Biosensors have been used in the determination of concentrations of various analytes in fluids for more than three decades. Of particular interest is the measurement of blood glucose, creatinine, creatine, and cholesterol.
It is well known that the concentration of blood glucose is extremely important for maintaining homeostasis. Products that measure fluctuations in a person""s blood sugar, or glucose levels, have become everyday necessities for many of the nation""s millions of diabetics. Because this disorder can cause dangerous anomalies in blood chemistry and is believed to be a contributor to vision loss and kidney failure, most diabetics need to test themselves periodically and adjust their glucose level accordingly, usually with insulin injections. If the concentration of blood glucose is below the normal range, patients can suffer from unconsciousness and lowered blood pressure that may even result in death. If the blood glucose concentration is higher than the normal range, the excess blood glucose can result in synthesis of fatty acids and cholesterol, and in diabetics, coma. Thus, the measurement of blood glucose levels has become a daily necessity for diabetic individuals who control their level of blood glucose by insulin therapy.
Patients who are insulin dependent are instructed by doctors to check their blood-sugar levels as often as four times a day. To accommodate a normal life style to the need of frequent monitoring of glucose levels, home blood glucose testing was made available with the development of reagent strips for whole blood testing.
One type of blood glucose biosensors is an enzyme electrode combined with a mediator compound that shuttles electrons between the enzyme and the electrode resulting in a measurable current signal when glucose is present. The most commonly used mediators are potassium ferricyanide, ferrocene and its derivatives, as well as other metal-complexes. Many sensors based on this type of electrode have been disclosed. Examples of this type of device are disclosed in the following patents.
U.S. Pat. No. 5,628,890 (1997, Carter et al.) discloses an electrode strip having an electrode support, a reference or counter electrode disposed on the support, a working electrode spaced from the reference or counter electrode on the support, a covering layer defining an enclosed space over the reference and working electrodes and having an aperture for receiving a sample into the enclosed space, and a plurality of mesh layers interposed in the enclosed space between the covering layer and the support. The covering layer has a sample application aperture spaced from the electrodes. The working electrode includes an enzyme capable of catalyzing a reaction involving a substrate for the enzyme and a mediator capable of transferring electrons between the enzyme-catalyzed reaction and the working electrode.
U.S. Pat. No. 5,708,247 (1998, McAleer et al.) discloses a disposable glucose test strip having a substrate, a reference electrode, a working electrode, and a means for making an electrical connection. The working electrode has a conductive base layer and a coating layer disposed over the conductive base layer. The coating layer is a filler having both hydrophobic and hydrophilic surface regions that form a network, an enzyme and a mediator.
U.S. Pat. No. 5,682,884 (1997, Hill et al.) discloses a strip electrode with screen printing. The strip has an elongated support that includes a first and second conductor each extending along the support. An active electrode, positioned to contact the liquid mixture and the first conductor, has a deposit of an enzyme capable of catalyzing a reaction and an electron mediator. A reference electrode is positioned to contact the mixture and the second conductor.
U.S. Pat. No. 5,762,770 (1998, Pritchard et al.) discloses an electrochemical biosensor test strip that has a minimum volume blood sample requirement of about 9 microliters. The test strip has a working and counter electrodes that are substantially the same size and made of the same electrically conducting material placed on a first insulating substrate. Overlaying the electrodes is a second insulating substrate that includes a cutout portion that forms a reagent well. The cutout portion exposes a smaller area of the counter electrode than the working electrode. A reagent for analysis of an analyte substantially covers the exposed areas of the working and counter electrodes in the reagent well. Overlaying the reagent well and affixed to the second insulating substrate is a spreading mesh that is impregnated with a surfactant.
U.S. Pat. No. 5,755,953 (1998, Henning et al.) discloses a reduced-interference biosensor. The device generally comprises an electrode used to electrochemically measure the concentration of an analyte of interest in a solution. The device Includes a peroxidase enzyme covalently bound to microparticle carbon and retained in a matrix In intimate contact with the electrode. According to this disclosure, it is the enzyme/microparticle carbon of the device that provides a composition that displays little sensitivity to known interfering substances.
It is well known that creatinine is a waste product derived from creatine and excreted by the kidneys. The analytical determination of creatinine in urine, serum or plasma is a widely used and extremely Important test for renal dysfunction. Measurements of creatinine in serum or urine may also be used as indices in the diagnosis and treatment of other disorders such as muscular dystrophy and hypothyroidism. Thus, the creatinine assay has been a widely recognized as having vital medical significance. Further, dietary changes have little if any influence on the creatinine concentration in blood and urine. Although creatinine is primarily a waste product, and as such plays no important role in biochemical functions of the body, its chemical precursor, creatine, has a vital biochemical role. Creatine is a basic building block of creatine phosphate, which is the primary form of energy storage in muscle. As a result, the creatinine level is an important diagnostic index for renal, muscular and thyroid function.
Spectrophotometry has been conventionally employed for measuring creatinine. The presence and concentration of creatinine in the above-mentioned body fluids is most frequently determined by the Jaffe reaction. In this reaction, creatinine reacts with picric acid to produce a red color, a red tautomer of creatinine picrate. This method suffers from serious disadvantages including, but not limited to, the instability of alkaline picrate solutions and the concomitant necessity for preparing solutions as needed, interference from blood metabolites, the analytical time required to perform the method, and the lack of specificity.
Sensors have been developed for the detection of creatinine based on enzymatic cleavage of creatinine. Among them, electrochemical methods received particular attention. Rechnitz et. al. (T. Huvin and G. A. Rechnitz, Anal. Chem., 46 (1974) 246) used creatinine deiminase coupled with an ammonia electrode to measure ammonia produced by an enzymatic reaction. However, this potentiometric method seems of little usefulness due to serious interference problems and the sensitivity limitation of the gas-sensing electrode.
U.S. Pat. No. 5,958,786 (1999, C. Munkholm) provides for the coupling of the enzymatic cleavage of creatinine to detection by a fluorescent polymer coating. The polymer coating has a first layer of protonated pH sensitive fluorophore immobilized in a hydrophobic polymer. The fluorophore reacts quantitatively with ammonia. The transducing moiety of the fluorophore is neutrally charged when deprotonated. The polymer coating has a second layer of creatinine deiminase and a polymer, and a third layer of a polymer. A disadvantage of this device is that two consecutive readings must be made. First, a fluorescence measurement must be made of the creatinine sensor. Second, the sensor material of the creatinine sensor is then exposed to a solution containing creatinine followed by measuring the fluorescence change and determining the concentration of creatinine.
A more practical strategy was reported by Tsuchida and Yoda in 1983 (T. Tsuchida and K. Yoda, Clin. Chem., 2911 (1983) 51). The proposed system consisted of three enzymes, creatinine amidohydrolase (C1), creatine amidinohydrolase (C2) and sarcosine oxidase (SO). These enzymes were co-immobilized onto the porous side of a cellulose membrane. The membrane was combined with a polarographic electrode for sensing hydrogen peroxide, a product resulting from the enzymatic reaction. Several research groups attempted to improve electrode performance through better enzyme immobilization techniques. (H. Yamato, M. Ohwa and W. Wemet, Anal. Chem., 67 (1995) 2776; M. B. Madaras, I. C. Popescu, S. Ufer and R. P. Buck, Anal. Chim. Acta, 319 (1996) 335; J. Schneider, B. Grundig, R. Renneberg, K. Camman, M. B. Madaras, R. P. Buck and K. D. Vorlop, Anal. Chim. Acta, 325 (1996) 161). Despite the improvements in enzyme immobilization, the methods suffer from various shortcomings including long-term stability, appropriate dynamic measurement range and serious Interference from other oxidizable substances in the sample fluid such as ascorbic acid and acetaminophen as well as creatine.
Currently, two commercial products for measuring blood creatinine are available. One is from Nova Biomedical Corporation. It is a critical care analyzer that provides a complete 14-test profile from as little as 105 microliters of whole blood where one of the tests is for creatinine. The creatinine sensor is a multiple-use, membrane-based sensor arranged in a fluid channel along with other biosensors (Nova Stat Profile(copyright) M, Nova Biomedical Corporation, Waltham, Mass.). The enzymes are immobilized onto the membrane and the membrane is attached to the working electrode (platinum) and the reference electrode (Agxe2x80x94AgCl).
The second commercial product is from i-Stat Corporation (Kanata, Ontario, Canada). A US patent covers this product. U.S. Pat. No. 5,554,339 (1996, Cozzette et al.) discloses an amperometric base sensor fabricated on a planar silicon substrate by means of photolithography in combination with the plasma deposition of metallic substances. The metallic substances include iridium metal (used as working electrode) and silver metal (served as reference electrode along with resulting chloridized silver). Three enzymes (C1, C2 and SO) are immobilized onto the electrodes as an overlaid structure. The above two products require calibration before measurement and a relatively large amount of sample volume. They also require a relatively longer waiting time for test results.
Because of the significance of obtaining accurate analyte concentration measurements, it is highly desirable to develop a reliable, user-friendly and disposable sensor, which does not have all of the drawbacks previously mentioned. Therefore, what is needed is an electrochemical sensor that does not require routine maintenance. What is further needed is an improved electrochemical sensor that combines peroxidase with a mediator. What is still further needed is an improved electrochemical sensor that combines peroxidase with a mediator and that operates at a reductive potential where interferents are not oxidized. What is yet further needed is an improved creatinine electrochemical sensor that includes an interference-correcting electrode to minimize the interference effects caused by the presence of creatine in a sample fluid. What is yet further needed are improved electrochemical sensors for cholesterol, glucose and other biologically important metabolites. Yet, what is still further needed is an electrochemical sensor that requires less sample volume for measuring an analyte than previously required by the prior art. What is still further needed is an improved disposable sensor for self-testing.
It is an object of the present invention to provide an electrochemical sensor that does not require routine maintenance. It is a further object of the present invention to provide an electrochemical sensor that combines at least one enzyme with a peroxidase and a mediator. It is still a further object of the present invention to provide an electrochemical sensor that combines at least one enzyme with a peroxidase and a mediator and that operates at a lower potential where interferents are not oxidized. It is yet a further object of the present invention to provide a creatinine electrochemical sensor that includes an interference-correcting electrode to minimize the interference effects caused by the presence of creatine in a sample fluid. It is yet further object of the present invention to provide improved electrochemical sensors for cholesterol, glucose and other biologically important metabolites. It is yet another object of the present Invention to provide an electrochemical sensor with high sensitivity to the analytes to be measured. It is yet still a further object of the present invention to provide an electrochemical sensor that requires less sample volume for measuring analytes than previously required by the prior art. It is still a further object of the present invention to provide an improved disposable sensor for self-testing.
The present invention achieves these and other objectives by providing a simple and convenient method of measuring various analytes in biological fluids. Although the following describes a preferred design of the present invention, a sensor of the present invention may have different physical shapes without detracting from the unique characteristics of the present invention. The present invention has a laminated, elongated body having a sample fluid channel connected between an opening on one end of the laminated body and a vent hole spaced from the opening. Within the fluid channel lies one or more working electrodes and a reference electrode, depending on the analyte to be measured. The arrangement of the one or more working electrodes and the reference electrode is not important for purposes of the results obtained from the sensor. The working electrodes and the reference electrode are each in electrical contact with separate conductive conduits, respectively. The separate conductive conduits terminate and are exposed for making an electrical connection to a reading device on the end opposite the open channel end of the laminated body.
The laminated body has a base insulating layer made from a plastic material. Several conductive conduits are delineated on the base insulating layer. The conductive conduits may be deposited on the insulating layer by screen printing, by vapor deposition, or by any method that provides for a conductive layer that adheres to the base insulating layer. The conductive conduits may be individually disposed on the insulating layer, or a conductive layer may be disposed on the insulating layer followed by etching/scribing the required number of conductive conduits. The etching process may be accomplished chemically, by mechanically scribing lines in the conductive layer, by using a laser to scribe the conductive layer into separate conductive conduits, or by any means that will cause a break between and among the separate conductive conduits required by the present invention. Conductive coatings or layers that may be used are coatings of copper, gold, tin oxide/gold, palladium, other noble metals or their oxides,or carbon film compositions. The preferred conductive coatings are gold film or a tin oxide/gold film composition.
It should be pointed out that although the same electrically conducting substance (gold film or tin oxide/gold film) after scoring is used as conducting material for both the one or more working electrodes and the reference electrode, this material itself cannot function as a reference electrode. To make the reference electrode work, there must be a redox reaction (e.g., Fe(CN)63xe2x88x92+exe2x88x92⇄Fe(CN)64xe2x88x92 or AgCl+exe2x88x92⇄Ag+Clxe2x88x92) at the electrically conducting material when a potential is applied. Therefore, a redox reaction must be present at the conducting material used for the reference electrode.
In one embodiment of the present invention, the laminated body has a first middle insulating layer, also called a reagent holding layer, on top of the base insulating layer and the conductive conduits. The first middle layer, or reagent holding layer, contains cutouts for one or more working electrodes and a reference electrode. Each cutout corresponds to and exposes a small portion of a single conductive conduit. The cutouts for the working electrodes are substantially the same size. The cutout for the reference electrode may be the same or different size as the cutouts for the working electrodes. The placement of all of the cutouts are such that they will all co-exist within the sample fluid channel described above. This first middle insulating layer is also made of an insulating dielectric material, preferably plastic, and may be made by die cutting the material mechanically or with a laser and then fastening the material to the base layer. An adhesive, such as a pressure-sensitive adhesive, may be used to secure the first middle Insulating layer to the base layer. Adhesion may also be accomplished by ultrasonically bonding the first middle layer to the base layer. The first middle insulating layer may also be made by screen printing the first middle insulating layer over the base layer.
Each cutout contains electrode material. The electrode material has a redox mediator and a peroxidase. The peroxidase may be from any source such as soybean (soybean peroxidase (SBP)) or horseradish root (horseradish root peroxidase (HRP)). For most analytes such as glucose and cholesterol, at least one of the cutouts contains the electrode material and an analyte-related enzyme forming an enzyme mix capable of catalyzing a reaction involving a substrate for the enzyme, e.g. glucose oxidase (GOD) for glucose. The redox mediator is capable of transferring electrons between the enzyme-catalyzed reactions and the working electrode.
For analytes having a substrate capable of undergoing similar reactions and causing an interference effect, a multiple enzyme mix may be required. Creatinine is one such analyte. Both creatinine and creatine exist in the blood. To measure the enzyme creatinine using the present invention, at least one xe2x80x9cworking electrodexe2x80x9d cutout contains the electrode material and two enzymes, e.g. creatine amidinohydrolase (C2) and sarcosine oxidase (SO), capable of catalyzing a reaction involving a substrate for the enzyme creatine. This measures the creatine level. A second cutout contains the electrode material and three enzymes, e.g. creatinine amidohydrolase (C1), creatine amidinohydrolase and sarcosine oxidase, capable of catalyzing a reaction involving a substrate for the enzyme creatinine. The difference in output of the two working electrodes represents the concentration of creatinine in the samples.
The enzymatic-reaction sequence for a creatinine sensor is:                     Creatinine        +                              H            2                    ⁢                      O            ⁢                          ⟶              C1                        ⁢            Creatine                                              Eq        .                  xe2x80x83                ⁢                  (          1          )                                        Creatine        +                              H            2                    ⁢                      O            ⁢                          ⟶              C2                        ⁢            Sarcosine                          +        Urea                            Eq        .                  xe2x80x83                ⁢                  (          2          )                                        Sarcosine        +                              H            2                    ⁢          O                +                              O            2                    ⁢                      ⟶            SO                    ⁢          Glycine                +        HCHO        +                              H            2                    ⁢                      O            2                                              Eq        .                  xe2x80x83                ⁢                  (          3          )                    
Creatinine measurements in the prior art are based on the amperometric detection of H2O2 resulting from the above enzymatic reaction. The enzymatic-reaction sequence for a glucose sensor is:                     Glucose        +                              H            2                    ⁢          O                +                                            O              2                        ⁢                          ⟶              GOD                        ⁢            Gluconic                    ⁢                      xe2x80x83                    ⁢          acid                +                              H            2                    ⁢                      O            2                                              Eq        .                  xe2x80x83                ⁢                  (          4          )                    
The present invention increases the sensitivity of the analyte measurement by incorporating a mediator and a peroxidase enzyme in the electrode material. The preferable mediators are redox chemicals either in oxidized or reduced form. The mediator used in the present invention may be at least one of a variety of chemicals in their reduced form, or virtually any oxidizable species or electron donors. Examples of useable compounds are Fe(CN)63xe2x88x92, Fe(CN)64xe2x88x92, Fe(phen)32+ (phen=1,10-phenanthroline), Fe(bpy)32+ (bpy=2,2xe2x80x2-bipyridine), Co(NH3)62+, Co(phen)32+, Co(bpy)32+, Os(bpy)2Cl+, Os(phen)2Cl+ Ru(bpy)22+, Rh(bpy)22+, cobalt phthalocyanine, various ferrocenes, methylene blue, methylene green, 7,7,8,8-tetracyanoquinodimethane (TCNQ), tetrathiafulvalene (TTF), toluidine blue, meldola blue, N-methylphenazine methosulfate, phenyldiamines, 3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzidine (TMB), pyrogallol, and benzoquinone (BQ). It is desirable that the mediator is capable of being oxidized chemically by hydrogen peroxide resulting from the enzymatic reactions such as those illustrated in Eqs. (1) to (3) and Eq. (4) above. It is further desirable that the oxidation form of the mediator is capable of being reduced electrochemically at the working electrodes at the applied potential. It is still further desirable that the mediator is stable in the matrix. The preferred mediator in the present invention is potassium ferrocyanide (K4Fe(CN)6).
The reduced form of the ferrocyanide mediator (Fe(CN)64xe2x88x92) is capable of being oxidized by the hydrogen peroxide resulting from the above enzymatic reaction to Fe(CN)63xe2x88x92 in the presence of a peroxidase. When using ferrocyanide as the mediator, the oxidation reaction is as shown below:                                           Fe            ⁡                          (              CN              )                                6                      4            -                          +                              H            2                    ⁢                                    O              2                        ⁢                          ⟶              SBP                        ⁢                                          Fe                ⁡                                  (                  CN                  )                                            6                              3                -                                                    +                              H            2                    ⁢          O                                    Eq        .                  xe2x80x83                ⁢                  (          5          )                    
The oxidized form of the ferrocyanide radical (Fe(CN)63xe2x88x92 is capable of being reduced electrochemically when a low potential is applied to the working electrodes. The resulting current signal is related to the analyte concentration.
It is well known that dissolved oxygen could be reduced at the electrode when a low potential is applied. Thus, it is desirable to apply a potential between the working electrodes and the reference electrode such that (Fe(CN)63xe2x88x92 is electro-reduced but dissolved oxygen is not or minimized. Furthermore, it is also desirable to use a potential where the electro-oxidation of other oxidizable interferents like ascorbic acid and acetaminophen either does not occur or is minimal. An example of such an applied potential is between about 0.0 V and about xe2x88x920.6 V as measured against the reference electrode of the present invention. The preferred potential is about xe2x88x920.15 V. This potential is preferred for providing a good ratio of signal vs. background noise/interference.
It is also desirable to minimize the interference from hematocrit (volume fraction of erythrocytes) on the results. Because the conductivity (or impedance) of whole blood is dependent on hematocrit, it can then be used to correct the effect of hematocrit on the reported concentration.
The resistance (r-value) between W (working electrode) and R (reference electrode) is related to the hematocrit as representated by the following equation:
r=k1/(1xe2x88x92H)xe2x80x83xe2x80x83Eq. (6)
where r is resistance value measured in Ohms or Kilo-Ohms
H is hematocrit level
k1 is a constant (r measured in Kilo-Ohms)
The measured xe2x80x9crxe2x80x9d can then be used to correct the analyte concentration. The relationship is represented by the Equation (7).:
Ccorr =k2xc3x97Cmeaxc3x97r/r0xe2x80x83xe2x80x83Eq. (7)
where Ccorr is the corrected analyte concentration
Cmea is the measured analyte concentration
r0 is the resistance value in Ohms or Kilo-Ohms measured at a preselected normal hematocrit
k2 is a constant
The laminated body also has a second middle insulating layer, also called a channel-forming layer, on top of the first middle layer. The second middle layer, or channel-forming layer is also made of a plastic insulating material and creates the sample fluid channel of the laminated body. It contains a U-shaped cutout on one end which overlays the cutouts on the first middle layer with the open end corresponding to the open end of the laminated body described earlier.
The laminated body of the present invention has a top layer with a vent opening. The vent opening is located such that at least a portion of the vent opening overlays the bottom of the U-shaped cutout of the second middle insulating layer. The vent allows air within the sample fluid channel to escape as the sample fluid enters the open end of the laminated body. The sample fluid generally fills the sample fluid channel by capillary action. In small volume situations, the extent of capillary action is dependent on the hydrophobic/hydrophilic nature of the surfaces in contact with the fluid undergoing capillary action. This is also known as the wetability of the material. Capillary forces are enhanced by either using a hydrophilic insulating material to form the top layer, or by coating at least a portion of one side of a hydrophobic insulating material with a hydrophilic substance in the area of the top layer that faces the sample fluid channel between the open end of the laminated body and the vent opening of the top layer. It should be understood that an entire side of the top layer may be coated with the hydrophilic substance and then bonded to the second middle layer.
The insulating layers of the laminated body may be made from any dielectric material. The preferred material is a plastic material. Examples of acceptable compositions for use as the dielectric material are polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane, cellulose nitrate, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, acrylic, and polystyrene.
In a second embodiment of the present invention, a first middle layer is not required for those analyte-measuring electrode systems where there are no competing substrate reactions for the enzyme. In other words, where there is no need for a second working electrode such as in the creatinine measuring system of the present invention.
In the embodiments using a first insulating layer, two cutouts contain material for the working electrodes (W1 and W2) and one for the reference electrode (R). The positional arrangement of the two working electrodes and the reference electrode in the channel are not critical for obtaining useable results from the electrochemical sensor. The possible electrode arrangements within the sample fluid channel may be W1-W2-R, W1-R-W2, R-W1-W2, W2-W1-R, W2-R-W1, or R-W2-W1 with the arrangement listed as the arrangement of electrodes would appear from the open end of the laminated body to the vent opening. The preferred position was found to be W1-R-W2; that is, as the sample fluid entered the open end of the laminated body, the fluid would cover W1 first, then R, then W2. The working electrodes and the reference electrode are each in electric contact with separate conductive conduits, respectively. The separate conductive conduits terminate and are exposed for making an electric connection to a reading device on the end opposite the open channel end of the laminated body.
In the creatinine sensor, the first working electrode (W1) is loaded with a mixture of C2, SO, a peroxidase, potassium ferrocyanide, at least one binder, and a surfactant. The second working electrode (W2) is loaded with the same chemical reagent as W1 but with the addition of C1. The reference electrode (R) cutout is loaded with a mixture containing at least one of the redox mediators mentioned above, at least one binder, and a surfactant. It should be noted that W1 is substantially a creatine sensor, while W2 is substantially a sensor responding to creatinine and to creatine. The difference between the electrode responses at W2 and W1 corresponds to the creatinine concentration.
In a glucose sensor, the first working electrode is loaded with a mixture of glucose oxidase, a peroxidase, potassium ferrocyanide, at least one binder, and a surfactant. In a cholesterol sensor, the first working electrode is loaded with a mixture of cholesterol esterase, cholesterol oxidase, a peroxidase, potassium ferrocyanide, at least one binder, and a surfactant. The reference electrode may be loaded with the same mixture as the working electrode. It should be pointed out that the reference electrode cutout could be loaded with a Ag/AgCl layer (e.g. by applying Ag/AgCl ink or by sputter-coating a Ag or Ag/AgCl layer) or other reference electrode materials instead of a redox mediator.
As mentioned earlier, oxidizable interferents such as ascorbic acid, uric acid and acetaminophen, to name a few, cause inaccurate readings in the output of an electrochemical biosensor. The present invention reduces this effect considerably by using an applied potential that minimizes oxidaton of these interferents. Also important is the composition of the reagents disposed on W1 and W2. The reagents are designed to have a minimal effect on the response of the interferences which also contributes to the accuracy of the analyte measurement.
All of the advantages of the present invention will be made clearer upon review of the detailed description, drawings and appended claims.